1. Field of the Invention
This invention relates to detection and measurement of FK778 or analogues of FK778 in biological samples, and, more particularly, detection and measurement of FK778 or analogues of FK778 in biological samples by means of competitive immunoassay.
2. Discussion of the Art
FK778 (previously known as HMR 1715, X92 0715, or MNA 715) is structurally similar to A77 1726, the active metabolite of leflunomide. Like A77 1726, FK778 is a malononitrilamide; these compounds are effective immunosuppressants in experimental models of autoimmune diseases and in allo- or xeno-transplantation. While leflunomide (Arava™) has been released for clinical use in rheumatoid arthritis, the long plasma half-life of A77 1726 in humans (15-18 days) makes the drug undesirable for use in clinical transplantation. FK778 has a shorter plasma half-life in humans and thus holds promise that it might be useful in clinical transplantation. The structures of FK778 and A77 1726 are set forth below.

FK778 is described in greater detail in the following articles, all of which are incorporated herein by reference:    Bilolo et al., “SYNERGISTIC EFFECTS OF MALONONITRILIAMIDES (FK778, FK779) WITH TACROLIMUS (FK506) IN PREVENTION OF ACUTE HEART AND KIDNEY ALLOGRAFT REJECTION AND REVERSAL OF ONGOING HEART ALLOGRAFT REJECTION IN THE RAT”, Transplantation, Vol. 75, 1881-1887, No. 11, Jun. 15, 2003.    Birsan et al., “In vivo pharmacokinetic and pharmacodynamic evaluation of malononitrilamide FK778 in non-human primates”, Transpl. Int. (2003) 16: 354-360.    Birsan et al., “Effects of the malononitrilamide FK778 on immune functions in vitro in whole blood from non-human primates and healthy human volunteers”, Transplant Immunology 11 (2003) 163-167.    Fawcett et al., “FK778: A Powerful Immunosuppressive, But Will It Really Be Good for You?”, Transplantation, Volume 78, Number 1, Jul. 15, 2004.    Evers et al., “Inhibition of human cytomegalovirus signaling and replication by the immunosuppressant FK778”, Antiviral Research xxx (2004) xxx-xxx.    First et al., “NEW DRUGS TO IMPROVE TRANSPLANT OUTCOMES”, Transplantation, Vol. 77, S88-S92, No. 9, May 15, 2004 Supplement.    Jin et al., “A novel leflunomide derivative, FK778, for immunosuppression after kidney transplantation in dogs”, Surgery, Volume 132, Number 1, 72-79, July 2002.    Savikko et al., “Leflunomide Analogue FK778 Is Vasculoprotective independent of its Immunosuppressive effect: Potential Application for Restenosis and Chronic Rejection”, Transplantation 2003; 76: 455, Transplantation, Vol. 76, 471-473, No. 3, Aug. 15, 2003.    Savikko et al., “LEFLUNOMIDE ANALOGUE FK778 IS VASCULOPROTECTIVE INDEPENDENT OF ITS IMMUNOSUPPRESSIVE EFFECT: POTENTIAL APPLICATIONS FOR RESTENOSIS AND CHRONIC REJECTION”, Transplantation, Vol. 76, 455-458, No. 3, Aug. 15, 2003.    Slauson et al., “Flow cytometric analysis of the molecular mechanisms of immunosuppressive action of the active metabolite of leflunomide and its malononitrilamide analogues in a novel whole blood assay”, Immunology letters 67 (1999) 179-183.    Vanrenterghem et al., “The Effects of FK778 in Combination With Tacrolimus and Steroids: A Phase II Multicenter Study in Renal Transplant Patients”, Transplantation, Volume 78, Number 1, Jul. 15, 2004.
Detection and measurement of FK778 in biological samples is important for monitoring therapeutic drugs, as an aid in adjusting drug dosage. The concentration of drug in plasma correlates to the degree of immunosuppression.
FK778 can be determined by LC/Tandem Mass Spectrometry. Methods for LC/Tandem Mass Spectrometry are described in the following reference, which is incorporated herein by reference:    Therapeutic Drug Monitoring of Immunosuppressant Drugs by High-performance Liquid Chromatography-Mass Spectrometry. Taylor, Paul J. Therapeutic Drug Monitoring 26(2):215-219, April 2004
Determination of the presence and amount of FK778 or analogues of FK778 in a biological sample can be determined by a competitive diagnostic assay. Small molecule, competitive diagnostic assays usually require a labeled component that can compete with the analyte for available antibody sites. The labeled component is typically referred to as a tracer. Examples of the labeled component include radioactive tracers, fluorescent tracers, chemiluminescent tracers, and enzyme tracers. Typically, the labeled component consists of the analyte or an analogue of the analyte coupled to a label.
The probability that a particular reagent comprising an antibody to FK778 and a labeled component will be useful in a sensitive assay for FK778 can be assessed by knowledge of the dose response curve. The dose response curve for a FK778 assay is a plot of the ratio of the response in the presence of FK778 analyte to the response in the absence of FK778 analyte as a function of the concentration of the FK778 analyte. The dose response curve for a given FK778 assay is unique for each reagent comprising an antibody to FK778 and a tracer and is modulated by the competition between the tracer and the analyte for sites on the antibody to the analyte.
The problem with a typical FK778 competitive immunoassay on an automated chemiluminescent analyzer is that the tracer comprising FK778 and an acridinium label has a very potent signal. Consequently, the tracer must be diluted to a very low concentration to be measured by the analyzer. FK778 analyte is present at a very high concentration in biological samples. Accordingly, the sample must be diluted more than 1000-fold to compete effectively with the tracer. This degree of sample dilution is typically not available on an automated analyzer. Failure to provide such a dilution results in an assay in which the concentration of FK778 exceeds the concentration of the tracer by so much that the dose response curve is too steep in the dynamic range for a reliable assay. It is desired to develop a competitive assay that allows effective competition between a tracer and the analyte but contains a labeled component that is not as potent as the tracer comprising FK778 and an acridinium label. It would be desirable to provide a competitive immunoassay format capable of detecting levels of FK778 above 10 μg/mL and below 250 μg/mL, concentrations that are clinically useful but difficult to measure.
The company that developed the FK778 drug for clinical use (formerly Fujisawa, now Astellas) also developed and evaluated a series of monoclonal antibodies to FK778 using an ELISA procedure. It was required that these antibodies have sufficient affinity for FK778 in order to be used in an immunoassay. In addition, even when antibodies that demonstrated an appropriate affinity for the FK778 analyte were developed, many of these antibodies demonstrated the undesirable property of high cross-reactivity to structurally similar analogues of FK778, such as metabolites. These antibodies were further screened by Fujisawa for degree of cross-reactivity to metabolites and an antibody having low cross-reactivity was selected.